Nectariferous plants biodiversity from different climatic and geographical areas of Croatia
(Pannonian, Mountain and Adriatic) offers a great potential for the production of specific
types of honey. The diversity that we have in climazonal and biocenological schedule
represents a unique national treasure that is in Europe rarely found. However, due to nonsystematic
research, pollen spectrum and chemical properties of most Croatian honeys
are poorly understood. One of the honey types which is not systematically researched is
mint (Mentha spp.). According to available literature on European unifloral honeys, mint
honey belongs to rare types. Therefore the aim of this study was to characterize the mint
honey through determination of botanical origin (pollen spectrum) and content of the
honey sac and certain physical and chemical parameters (water content, electrical
conductivity and pH value). The second goal was to determine the profile of volatile
compounds in the honey sac content, as well as the end product, honey. At the same
time, one of the study goals was to investigate the behaviour of bees on mint nectar flow
through the foraging activity of bee colonies.
The research was conducted at the experimental site Mahovo, which is located in the
Lonjsko Polje Nature Park. Five experimental colonies of Carniolan (Apis mellifera carnica
Pollmann, 1879) bees in standard Langstroth-Root (LR) colonies were located at the
selected location. Gathering activity of honey bee colonies was conducted by sampling
from each colony three times a day (9:00, 14:00 and 18:00) for six days. Monitoring of the
foraging on the mint field was conducted on the five separate plots of 1 m2 by counting
bees for 1 min on the either side of the plot three times a day (10:00, 13:00, and 18:00) for
30 minutes during six days. Analysis of the honey-sac content and pollen loads was
carried out according to the method of Soehngen and Jay (1974). Qualitative
melisopalinological analysis of collected samples of honey, honey-sac content and the
pollen loads was conducted according to the methodology of von der Ohe et al. (2004).
From the physicochemical analyses, water content, electrical conductivity and pH were
performed. Analysis of volatile compounds was performed using solvent-free headspace
solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) in the
honey samples and the honey sac content.
From the totally analysed 8 721 workers, 2 992 have brought nectar, pollen, or both, while
5 471 returned to the hive without load. From analysed foragers that delivered load, the
main portion was of those who have gathered nectar (1 774), then those who have
gathered pollen (1 190), but the least those with honey and pollen load, which implies that
most foragers are specialized in collecting either nectar or pollen. Statistically higher
number of foragers with nectar or pollen load were determined in the early afternoon
(14:00) compared with foragers collected in 9:00 and 18:00.
The average weight of honey-sac content ranged from 9.58 to 12.22 mg with a range of
0.20 to 50.60 mg. Significantly lower average weight of honey-sac content (9.02 mg) was
determined in collected samples of bees at 9:00 o’clock compared to samples collected at
18:00 (11.06 mg) and 14:00, when average weight of 12.04 mg was determined. In the
largest group of foragers weight load was ≤ 10 mg, followed with group with load from 11
to 20 mg, and at the least number of bees collected ≥ 31 mg load.
Average weight of pollen load was from 4.77 to 9.60 mg, with range of 0.10 to 31.70 mg.
In the group of foragers collected at 9:00 o’clock statistically lower pollen load weight (7.73
mg) was determined in comparison to samples collected at 14:00 (8.63 mg). Contrary,
between groups collected at 9:00 and 18:00 as well as at 14:00 and 18:00 o’clock
difference was not significant.
Regarding attendance of bees to mint flowers, statistically lowest number of bees was
determined at 18:00 o’clock (20.08) in comparison to count at 10:00 (30.22) and 13:00
knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of
the results, this study provides a contribution in practical terms, because they indicate
preliminary actions to be undertaken for the timely preparation of colonies for the main
pasture. At the same time, the original data on the profile of mint honey volatile
compounds can be used as potential biomarkers to determine its botanical origin.
Keywords: honeybee colony foraging activity, honey sac, pollen load, mint (Mentha spp.)
honey, pollen spectrum, volatile compounds
(36.50) o’clock.
According to pollen spectrum, accompanying plants to Mentha pulegium were Solidago
gigantea, Amorfa fruticosa, Castanea sativa and Lotus corniculatus.
From pollen loads of collected foragers it was determined that 37.82% samples contain
mint pollen, from which most part (20.42%) is from pennyroyal (M. pulegium) in
comparison to 1.93% of water mint (M. aquatica). Apart from mint pollen, additional 13
plant species were determined in the pollen loads, and the most frequent were Daucus
carota, Salvia spp, Ranunculus spp., Inula britanica, Plantago lanceolata, Solidago
gigantea, Amorfa fruticosa and Centaurea spp.
The average water content was 17.3% with a range from 14.5 to 18.8%. Electrical
conductivity was 0.62 mS/cm and the range from 0.37 to 0.75 mS/cm. pH value was in
range from 3.52 to 4.15 with the average of 3.80.
Based on the volatile compounds analysis of the honey-sac content, the most intensive
were 3 compounds: 3.7-dimethylocta-1.5-dien-3.7-diol (terpendiol I), followed by methyl
syringate and vomifoliol. In the extract of analysed honey samples were also 3.7dimethylocta-1.5-dien-3.7-diol
(terpendiol I). Also, by the analysis of major honey
headspace compounds hotrienol, cis- and trans-linalool oxides, linalool and neroloxide
were determined.
Valuable scientific contribution of the dissertation results from the first time connection of
the honey-sac content and the end product of honey, from monitoring of pollen spectrum
changes and the knowledge of volatile compounds fate. Analysis of mint honey was
performed using two complementary techniques, namely solvent-free headspace solidphase
microextraction (HS-SPME) and ultrasonic solvent extraction (USE). It was found
that both methods are suitable, since did not cause the formation of thermal artefacts
during the extraction of volatile compounds. Also, these methods are complementary
because the HS-SPME method enables extraction of the most volatile compounds which
are in equilibrium with a honey solution, while the USE method allows the isolation of
volatile and semi-volatile compounds. These methods are applied to mint honey for the
first time and enabled the preparation of chemical profiles, which represents an original
contribution to this work. The organic extract of the foragers’ honey sac content on mint
nectar flow was analysed for the first time and methyl-siringat, terpendiol I and vomifoliol
found that can be attributable to plant origin. The other major compounds analysed in the
honey sac content were close to the part of cuticular waxes and less to bee pheromones.
Methyl syrnigate was the most common compound in ultrasonic assisted organic honey
extracts together with vomifoliol. Comparison of the organic honey extracts and extracts of
the honey sac content indicated that the methyl-syringate and vomifoliol are transferred to
honey, while terpendiol was partially transformed into hotrienol during maturation of
honey, which is also the original contribution to knowledge about the transport of volatile
compounds in relation nectar - honey sac - honey. Also, study results, based on exact
data, represent scientific contribution related to the biology of honeybee colony, i.e.,
knowledge of the behaviour of bees foraging on mint. In addition to the scientific value of
the results, this study provides a contribution in practical terms, because they indicate
preliminary actions to be undertaken for the timely preparation of colonies for the main
pasture. At the same time, the original data on the profile of mint honey volatile
compounds can be used as potential biomarkers to determine its botanical origin.